Naked and calcifying cells of E. huxleyi (strain E. huxleyi, abbreviated N-E; strain E. huxleyi, CS369, abbreviated C-E) and G. oceanica (strain G. oceanica, NIES-1318, abbreviated N-G; strain G. oceanica, abbreviated C-G) were obtained from the Yellow Sea Fisheries Research Institute Microalgae Culture Center. Taxonomic relationships of the four coccolithophore strains are depicted in Fig. S1. The cells were cultured in filtered (0.2 µm) natural seawater enriched with nitrate and phosphate to concentrations of 882 and 36.2 µmolL-1, respectively, and with metals and vitamins according to f/2 (Guillard and Ryther, 1962) at 20 ∘C and 32 ‰ salinity. The cells were exposed to a light : dark cycle (16:08 h) and saturating light (300 µmol photons m-2s-1) provided by white fluorescent lighting in an incubator. The culturing was conducted in sterilized 2 L Erlenmeyer flasks containing 1 L of medium, and algae in the exponential growth phase were used for inoculation in the experiments.

We conducted replicate experiments by bubbling cultures of four species of coccolithophores using air with different CO2 partial pressures and adopting a flask and tubing system manipulated in plant growth chambers (GXZ, Ruihua, Wuhan, China) similar to Xu et al. (2014). The experiments were prepared with monospecific cultures of the four species of coccolithophore and reached the exponential growth phase under the above-mentioned conditions. Three replicate cultures of 1000 mL of each coccolithophore were incubated with aeration of different CO2 partial pressures in a CO2 chamber. All the experiments were generally similar in design. Differences are shown in Table S1, and treatments were bubbled continuously with air containing different partial pressures of CO2 to provide 380, 750, 1000 and 2000 µatmCO2 in the medium. The culture medium typically became stable after 48 h of bubbling and before the start of the experimental inoculation. In each experiment, triplicate flasks for each air-CO2 mixture were maintained without added cells as a blank. The cell density and pH were monitored, respectively. Triplicate sampling for culture medium, chlorophyll (Chl) content, PIC, POC, DIC and alkalinity measurements were conducted at the sampling point.

The corresponding pH of the culture medium was monitored with a pH meter (Orion ROSS, Thermo Electron Corp., Beverly, MA, USA). The samples for total alkalinity (TA) measurements were immediately filtered using a Whatman GF/F and stored in sealed 100 mL borosilicate bottles at -20 ∘C until measured. The TA value was obtained using the 848 Titrino plus automatic titrator (Metrohm, Riverview, FL, USA). Parameters for the seawater carbonate system within experiments were calculated using the CO2SYS Package based on the pH, temperature, salinity, and TA according to the method of Pelletier et al. (2007).

An accurate cell count was obtained to estimate microalgal growth by manual counting with a hemocytometer under an optical microscope (Nikon, Tokyo, Japan). The specific growth rate (μ) was determined as follows: μ=(ln⁡C1-ln⁡C)/(t1-t), where C and C1 are the cell concentrations at initial time t and subsequent time t1, respectively.

The photosynthetic characteristics of tested coccolithophores were simultaneously determined using the pulse–amplitude modulated method with a Dual-PAM-100 (Walz, Effeltrich, Germany) connected to a PC running WinControl software. Before measurement, the samples were kept in the dark for 15 min, and the original fluorescence (F) was determined under low light. A saturation light pulse was applied to obtain maximum fluorescence (Fm) in the dark-adapted samples. The Fm yield in the illuminated samples was denoted as Fm′, and Ft′ was the real-time fluorescence yield. The maximal PSII quantum yield (Fv/Fm) was calculated according to the following equation: fv/Fm=(Fm-F)/Fm. The effective PSII quantum yield was calculated as follows: Y(II)=(Fm′-Ft′)/Fm′.

To determine the pigment content, the cells were collected onto Whatman GF/F filters (25 mm) at every sampling point and then extracted in 10 mL of 90 % acetone for 24 h at 4 ∘C in darkness. After centrifugation at 7000×g for 10 min, the supernatant was used for content analysis at room temperature. The content of the chlorophyll was spectrophotometrically determined using the method of Jeffrey and Humphrey (1975).

Water samples (5 mL) from the experiment were collected and filtered quickly using acetate cellulose filters and then frozen at -80 ∘C in polyethylene flasks for storage until analysis. Before analysis, stored culture media samples were thawed to room temperature, and nitrate concentrations were analyzed photometrically using an AutoAnalyzer (BRAN and LUEBBE AA3, Germany). Nitrogen uptake rates were calculated as follows: NUR=(C-Ct)V/N/t, where NUR is the nitrogen uptake rate (pmol of nitrate cell-1 day-1); C and Ct are the nitrate concentrations (pmolL-1) at the beginning and on the 7th day of the experiment, respectively; V is the volume of the water (in L); N is the cell number (ind); and t is the time interval (in days). Moreover, we used Michaelis–Menten (Michaelis and Menten, 1913; Hutchins et al., 2013) rectangular hyperbolic saturation equation curves to fit CO2 response curves for the nitrogen uptake rates in each of the triplicate cultures and for each strain in each pCO2 treatment. The work was completed using OriginPro software, including the calculation of kinetic constants and curve correlation coefficients.

The samples for element analysis were taken from each replicate, filtered onto pre-combusted (500 ∘C for 5 h) Whatman GF/F filters (25 mm) and stored at -20 ∘C until analysis. Prior to analysis, the POC filters were fumed over HCl for 24 h to remove all inorganic carbon and then dried again. The filters were then packed in solvent-rinsed tin sample boats for analysis. The POC, PON and TPC (total particulate carbon) concentrations were determined sequentially with a Vario EL III automatic elemental analyzer (Elementar Analysensysteme Co., Germany). The PIC concentration was calculated by the differences between TPC and POC, and the POC, PIC or PON production was calculated as follows: P=specific growth rateμ(d-1)×cellular POC, PIC or PON content(pgcell-1).

All statistical analyses were performed using the SPSS (Statistical Package for the Social Sciences) 17.0 data processing system software, and the mean ±SD was calculated for each experiment. A one-way ANOVA was used to analyze the significance of variance among treatments. The significance level was set at 0.05 for all tests unless otherwise stated.

This experiment adopted a gas bubbling method to mimic seawater acidification and monitored the shift in parameters of the seawater-carbonate system, including pH, DIC, HCO3-, CO32-, and CO2, under different treatments. Figure S2 and Table S1 in the Supplement present the changes of seawater pH when culturing different isolates vs. different CO2 concentrations. The initial pH values corresponding to elevated pCO2 values of 380, 750, 1000 and 2000 µatm were 8.06±0.02, 7.79±0.01, 7.65±0.02 and 7.48±0.02, respectively. The experimental conditions set in this study were not exactly the same as those expected in ocean acidification because seawater contains buffers to induce changes in alkalinity. Some measurements required a large biomass; therefore, cell density was also very high, leading to a variation in the carbonate system. Moreover, although these shifts existed, a clear difference in pH among the four gradient cultures was maintained throughout the experiment. Therefore, the results are strongly supported and have significant implications.

The effect of different pCO2 on the growth of four coccolithophores in plant growth chambers is presented in Fig. 1. The cell concentration in all treatments increased gradually over time to a maximum, with a subsequent decline at the end of the experiment, and there was significant effect of pCO2 on cell concentration for every species. The C-E, N-E and N-G showed a positive response to gradually increased CO2 concentration, while the cell concentration of the N-E was suppressed as pCO2 reached 2000 µatm. Algal cells grew exponentially in all treatments during the first 7 days, and the specific growth rate is presented in Fig. 1e–h. Increasing the pCO2 by 2000 µatm increases the specific growth rate by approximately 53 % (the C-E), 36 % (the N-G) and 30 % (the C-G), respectively. However, when the bubbling CO2 concentration reached 1000 µatm, the specific growth rate increased by approximately 26 % for the N-E; with a continuous rise to 2000 µatm, the ocean acidification decreased the specific growth rate of the N-E by 14 % compared to the control. Moreover, when a comparison was conducted between or within species, the variability of E. huxleyi (naked strain to calcifying strain: reducing by 14 % to increasing by 53 %) exceeded that of the species G. oceanica (naked strain to calcifying strain: increasing by 36 and 30 %, respectively) under different CO2 concentrations.

Figure 2 shows the effects of ocean acidification by increasing pCO2 on the changes in the photosystem activity parameters, including Fv/Fm and Y(II), during the culture of coccolithophores. Both Fv/Fm and Y(II) in all treatments showed a negative influence from increasing pCO2 by 2000 µatm, but the magnitude was different among the four species. With incubation time, every photosystem activity parameter of the four coccolithophores simultaneously reached a maximum value on the 7th day, before a subsequent decline till the end of experiment, except for the parameter Fv/Fm of C-E (on the 10th day) and the N-E (continuous decline) for the 2000 µatm sample point. As pCO2 increased by 2000 µatm in plant growth chambers, significant suppression of the Fv/Fm from ocean acidification occurred on the 7th, 4th, 13th, and 10th day for the N-E, C-E, N-G, and C-G, respectively, and of the Y(II) on the 4th, 13th, 13th, and 10th day, respectively. At the end of experiment, the Fv/Fm was repressed by elevated pCO2 (2000 µatm) and decreased by 60, 45, 55, and 46 % and Y(II) also declined by 75, 69, 76, and 39 %, respectively, compared to the control. In addition, by comparison to the maximal Y(II) of N-E and N-G (under 380 µatm; 0.32±1.9 % and 0.34±2.6 %, respectively), the maximal Y(II) of the C-E and C-G (under 380 µatm; 0.39±3.1 % and 0.48±2.3 %, respectively) were significantly higher, while differences in Fv/Fm were not obvious among them.

Figure 3 presents the effects of elevated CO2 conditions from 380 to 2000 µatm on the intracellular chlorophyll content of different strains. After an initial value showing slight variation, the chlorophyll a content of cells increased to maximum values on the 20th (N-E, C-E, N-G) and 7th day (C-G), with a subsequent declining tendency until the final experiment (Fig. 3a–d). However, chlorophyll c displayed a steady overall decline over time (Fig. 3e–f). However, as the CO2 concentration increased from 380 to 1000 µatm, the chlorophyll content of every coccolithophore decreased in an orderly pattern (except for individual points in time that were presented differently) compared to 380 µatm at each sampling time. Nevertheless, the results determined under 2000 µatm conditions did not exhibit the same responses. During the 2000 µatmCO2 enrichment, the chlorophyll a and c content of the N-E with respect to cultivation time presented lower values than the 1000 µatmCO2 concentration at each sampling point. However, for the C-E, N-G and C-G strains, as the CO2 concentration in the culture increased above 1000 µatm, the chlorophyll content tended to increase at each sampling point.

The effect of ocean acidification on the nitrogen uptake rate of the four coccolithophores is displayed in Fig. 5. With increasing CO2 concentration from 380 to 2000 µatm, the nitrogen uptake rate of strains all increased, by 48.2 % (N-E), 33.9 % (C-E), 41.6 %(N-G) and 34.3 % (C-G). The maximum variation value (48.2 %, N-E) of the nitrogen uptake rate was nearly 1.5-fold larger than the minimum variation value (33.9 %, C-E). Moreover, the response of nitrogen uptake rates to increasing pCO2 in all the strains were closely described by a Michaelis–Menten curve fitting approach, and both half saturation constants (Km, µatmCO2) and maximum CO2-saturated rates (Vmax, pmol N cell-1 day-1) were obtained from the response curves. The maximum Km and Vmax value were 307.2 µatm and 24.1 pmolN cell-1 day-1 from strain N-E, respectively, which were approximately 1.7-fold and 1.3-fold larger than the minimum values presented by strain C-E, respectively.

Figure 5 shows the production of POC and PON of the four species of coccolithophore on the 7th day in elevated pCO2 conditions. Acidification by CO2 enrichment up to 2000 µatm distinctly stimulated the production of POC and PON of coccolithophores except for N-E , whose increasing POC production under 1000 µatm pCO2 conditions exhibited a significant decline at the 2000 µatm sample point, and the variations were enormous. For the C-E, N-G and C-G species, the POC production increased by approximately 101, 35 and 49 %, respectively, compared with the control, and the variation of the former reached 2.9 and 2.1-fold of the N-G and C-G, respectively. Moreover, over this range, PON production had a comparatively larger increase of 233 % (N-E), 289 % (C-E), 148 % (N-G) and 129 % (C-G) than controls. However, the changes of particle carbon and nitrogen production led to a decrease in the POC : PON for the four species by 76.6 % (N-E), 48.3 % (C-E), 45.7 % (N-G) and 34.9 % (C-G), with the pCO2 variation between 380 and 2000 µatm. In addition, the seawater acidification under 2000 µatm also had a negative effect on PIC production, which decreased by 35.4 and 68.9 % for two calcification species, C-E and C-G, respectively, in comparison with the production value at 380 µatm (Fig. 6). The C-G species presented a higher reduction ratio (approximately 1.9-fold) of PIC than the C-E when CO2 concentrations increased by 2000 µatm. Simultaneously, the ratio of PIC : POC for the two species also declined by 67.9 and 79.2 %.

This study showed that a high CO2 concentration (increasing to 2000 µatm) led to an increase in growth, except for N-E, whose growth declined at the 2000 µatm sample point and whose specific growth rates were significantly reduced by approximately 7.61 % on the 7th day in comparison to the control (Fig. 1). However, before pCO2 was elevated to 1000 µatm, the growth of N-E maintained an increasing state (Fig. 1a and e). Recently, Fiorini et al. (2011a) displayed similar results and showed an increasing growth trend of coccolithophores in CO2-rich water up to approximately 750 µatm.

Previous studies suggested that coccolithophores were carbon limited because of a comparatively less-efficient CO2 concentrating mechanism (CCM) caused by low activity for extracellular carbonic anhydrase in the present ocean (Herfort et al., 2002; Trimborn et al., 2007; Rokitta and Rost, 2012; Jin et al., 2013). Therefore, an increase in available CO2 by ocean acidification in seawater may accelerate the accumulation of CO2 in the vicinity of Rubisco to offset the short supply and further promote coccolithophore carboxylation and growth (along with an increase in POC) (Barcelose Ramos et al., 2010; Reinfelder, 2011; Jin et al., 2013; Kottmeier et al., 2014). In this study, we observed the increased growth of four strains including N-E (1000 µatm; 23 %), C-E (2000 µatm; 53 %), N-G (2000 µatm; 36 %) and C-G (2000 µatm; 30 %) (Fig. 1). However, every strain revealed different response capacities and strategies to the increasing pCO2. In addition, N-E was also significantly and negatively affected, which tended to lead to bleaching as the pCO2 value reached 2000 µatm (Fig. 1).

Recent studies reported that changes in extracellular pH could affect the activity of sensitive algal cells by upsetting the constant membrane potential balance and physiological parameters (Langer et al., 2006; Taylor et al., 2011; Rokitta et al., 2012). Hence, the more sensitive alga N-E, relative to the other three stains, showed a negative response because of the overstepping of its tolerance ability and declined pH (7.48, 2000 µatm, Table S1). Because coccolithophores are an abundant formal species, which have been characterized with a unique physiology and morphology (Westbroek et al., 1993; Winter and Siesser, 1994; Raven and Crawfurd, 2012), we propose that the observed differences in the response of coccolithophores to increasing pCO2 have a genetic basis. Recently, Read et al. (2013) reported the first haptophyte reference genome (from E. huxleyi CCMP1516) and assumed that genome variability within this species complex seems to underpin its capacity to thrive in different habitats. The inherent difference in adaptive capacity from coccolithophores would contribute to an inherent discrepancy reaction in response to ocean acidification, similar to the specific response results in this study.

However, algal photosynthesis (which is closely related to growth) that is evaluated by the activities of the photosystems and pigment content exerted a variable negative response with elevated pCO2, especially for N-E, which tended to bleach early in the experiment (Figs. 2 and 3). These results involving the activities of the photosystems revealed that cells remained “photosynthetically unhealthy” due to the effect on cell ion balance from declining pH (elevated pCO2) (Langer et al., 2006), which induced a decreasing in chlorophyll content (Fig. 3). In particular, N-E was seriously affected in comparison to the other coccolithophores when ocean acidification reached the 2000 µatm sample point, which showed exceeding maladjustment (Figs. 2a and 3a). Similar to the different responses in growth results with elevated pCO2, algal photosynthesis also reflected an inherent difference in its reaction. For the whole ecosystem, for which a slight change in one part may affect the whole situation, the specific reaction of coccolithophores to ocean acidification would likely affect biodiversity and other ecological process (Orr et al., 2005; Hendriks et al., 2010).

Considering the effect of ocean acidification on nutrient uptake, we tested the nitrogen uptake efficiency of each strain under different pCO2 levels and fitted nitrogen uptake rate response curves to changing pCO2 for further analysis at the 7th day sampling point. The results showed that the nitrogen uptake rate of all strains increased at the 2000 µatmCO2 concentration (Fig. 4). The naked strains N-E and N-G exhibited a greater increase of 48.2 and 41.6 % (Fig. 4a and c), respectively, while the nitrogen uptake rate increased by 33.9 and 34.3 % in the calcifying strain C-E and C-G, respectively (Fig. 4b and d). Jin et al. (2013) reported that inorganic nitrogen uptake was enhanced in coccolithophores and that the gene for nitrate reductase in a diatom was upregulated under elevated CO2 levels. In addition, disparities between strains also existed.

Previous studies suggested that coccolithophore cells exerted a process of energy redistribution under ocean acidification conditions (Raven, 2011; Beaufort et al., 2011), resulting in the release of extra ATP to preferentially support additional N uptake for the synthesis of more proteins (Jin et al., 2013). However, calcifying cells, which need more energy input for H+ from calcification, because there is an outward transport to acidic seawater or neutralization (Suffrian et al., 2011; Taylor et al., 2011; Beaufort et al., 2011; Stojkovic et al., 2013), absorbed substantially less nitrogen than naked strains for elevated pCO2 at 2000 µatm. In addition, the increasing PON production with elevated pCO2 determined in this study also indirectly confirmed the energy redistribution preference for nitrogen uptake (Fig. 5b).

The response curves of nitrogen uptake efficiency to changing pCO2 confirmed the influencing behavior of CO2 for each of these coccolithophores. Kinetic rate constants (Km value), which were derived from curves, represented the enzyme affinity to the substrate, and when the Km value increased, the affinity became smaller (Michaelis and Menten, 1913). Recently, Hutchins et al. (2013) documented strain-specific differences in the relationship between nitrogen fixation and CO2 concentration through different kinetic constant analysis. In this study, N-E showed the maximum Km value, namely, the minimum affinity, compared with other three strains (differences also existed among them) (Fig. 4). This result reflected the same trend with the other parametric measurements, such as growth rate, photosynthetic activity, etc., which indicated that N-E was seriously affected by elevated CO2 concentrations, and inter- and intra-specific responses were also displayed. In addition, the principal coordinate analysis (PCoA) and hierarchical clustering multi-variate statistical analyses for the replicates of each of the four coccolithophore strains displayed obvious differences in CO2 responses (Fig. S3). Replicates of each of the four strains formed well-defined clusters through PCoA (Fig. S3a). Moreover, independent hierarchical clustering of each replicate also showed the same serious groups, and this analysis revealed that the two calcifying strains were more similar to one another than they were to the two naked strains (Fig. S3b). These data analyses further highlighted the inter- and intra-specific variability in coccolithophore response to CO2-induced acidification.

Variations in the elemental stoichiometry of phytoplankton are known to have an effect on trophic interactions and ultimately exert an influence on marine nutrient biogeochemistry because the dietary value of prey for marine zooplankton varied with the POC to PON (C : N) ratio (Hutchins et al., 2009; Anderson et al., 2013). In this study, we measured the ratios of C : N to assess whether the elemental composition of the organic material was additionally affected by changing pCO2, and we also explored the extent of the impact. According to the results, changes in the elemental composition of coccolithophores grown at 2000 µatm pCO2 have been found (N-E, C-E, N-G and C-G), revealing a reduced C : N ratio (by approximately 76, 48, 45 and 35 %, respectively) (Fig. 5c). The ratio of N-E was affected greatly with respect to the other three isolates, and C-G was the least affected. In previous studies, similar variable ratios for coccolithophores based on an enhanced CO2 concentration were reported by Fiorini et al. (2011a, b) and Rickaby et al. (2010). These changes occurred with increasing POC (except for N-E at 2000 µatm of CO2) and PON production and under rising pCO2 concentration between 380 and 2000 µatm (Fig. 5).

It was also discovered that the incremental ratios of PON were higher than those of POC compared to the control groups with the ultimate proportion. We have discussed in the above section that the redistribution of energy resulted in more nitrogen absorption and increasing PON production with elevated pCO2. This preference advantage made the PON production exceed the POC production, even though differences existed in different strains, leading to different decreasing C : N ratios (Fig. 4c). Similar to previous research by Riebesell and Tortell (2011), the changes with which high pCO2 levels were associated with C : N ratios were highly species specific. Additionally, a variable proportion was also the embodiment of the inherited differences among different strains. In contrast, differences from current findings also exist, which show increased cellular C : N ratios with elevated pCO2 (Feng et al., 2008; De Bodt et al., 2010; Kottmeier et al., 2014).

The reasons for the opposite result may be a difference between the experimental settings, such as temperature, illumination intensity, and/or choice of algae strains. In addition, the final results exhibited a very interesting phenomenon, that is, that the reduced C : N ratios of the lost calcification strain were higher than those of the calcification strain in each species. The reason for the phenomenon is still unclear, but it was most likely associated with shell calcification. This part of the observed results indicated that the increased pCO2 affected algal C : N ratios and produced a diversity of effects on different coccolithophores. In addition, it will further influence grazing-selection pressure on phytoplankton and has many biogeochemical consequences (in particular implications for the export flux of carbon) (Iglesias-Rodriguez et al., 2008).

In the past few years, PIC production (calcification efficiency) has been widely investigated in coccolithophores to predict the impact of ocean acidification, and results show that the predominant response was a decreased rate of calcification when cells were grown at CO2 levels higher than those found today (380 µatm) or at least a decrease in PIC : POC (e.g., Zon-dervan et al., 2001; Langer et al., 2006; Muller et al., 2010; Hoppe et al., 2011). In this research, the response regarding PIC was in agreement with the response already described in the above-mentioned tendency. Namely, calcification species E. huxleyi and G. oceanica experienced a comparatively larger decrease in the rate of calcification of 35.4 and 68.9 %, respectively, between 380 and 2000 µatm at the 7th day sampling point (Fig. 6). In comparison, G. oceanica was more vulnerable than E. huxleyi under ocean acidification, leading to an unbalanced ecological factor. However, regarding lost calcification species in the experiment, the PIC production was nearly negligible because of the lost calcification ability to cover shells (not show).

Recent studies with various organisms showed calcification to be largely controlled by Ω-cal rather than by pH alone (Langer et al., 2006; Trimborn, 2007), and Ω-cal was controlled by both [DIC] and pH (Iglesias-Rodriguez et al., 2008). When supersaturated surface seawater (i.e., Ω>1) of calcium carbonate minerals (which was considered generally less than current ocean conditions, Feely et al., 2009) declined in saturation (Ω=1) because of ocean acidification or other natural processes, carbonate biominerals in shells and skeletons may begin to dissolve (Feely et al., 2010). The data in this study showed that Ω-cal ranged from 7.76 (C-E) and 8.79 (C-G) at 380 µatm of pCO2 to 1.58 and 1.57 at 2000 µatm of pCO2 (Fig. S4), which were above the threshold at which dissolution occurs. Nevertheless, the results clearly revealed that PIC production (calcification efficiency) finally had declined in comparison with the control group (Fig. 6).

From the results reported by Langer et al. (2009), the same phenomenon was also observed when Ω-cal was above 1, and dissolution still occurred. To explain this phenomenon, we found that the pH values of the cultures incubated at 380 and 2000 µatm of CO2 ranged between 8.81 and 7.62 on the 7th day (Table S1 and Fig. S2), and a shift in pH was caused by comparing blanks with cell physiology. These changes affected the photosynthetic health of cells, as discussed above, which implies that our pH conditions were not completely within the tolerance levels of cells. Bach et al. (2011) suggested that calcification is specifically responsive to the associated decrease in pH. Under elevated pCO2 circumstances, coccolithophore calcification, which is a stringently controlled biological process (Mackinder et al., 2010), would be affected by a decreasing pH level.

Recently, substantial physiological and molecular evidence has indicated that when HCO3- is used for calcification (which produces H+ and also maintains a cytosolic pH homeostasis near neutrality), the H+ must be removed across the plasmalemma H+ channel (Suffrian et al., 2011; Taylor et al., 2011) or neutralized in coccolithophores cells (Fabry et al., 2008; Rokitta and Rost, 2012; Stojkovic et al., 2013). To maintain the appropriate transplasmalemma electrical potential difference and the H+ efflux, energy input is needed in the process of H+ transport (Raven, 2011). When elevated pCO2 resulted in a low pH and/or [CO32-] level in waters, this input was likely greater per unit calcification and led to decreased calcification rates (Raven et al., 2011; Beaufort et al., 2011). Based on research of the adverse impact caused by increasing pCO2 on coccolithophore calcification, which was associated closely with biogeochemical cycles, although we give a reasonable explanation for decreasing calcification, this explanation is only a small fraction of the truth. Currently, the reasons for the impacts of ocean acidification on calcified coccolithophores still need further exploration.

Through our research, different coccolithophores revealed synthetically inter- and intra-specific variability to cope with the threat from ocean acidification and showed a significantly different response derived from inherent heredity. With the CO2 concentration gradual increasing, the original state maintained steadily by different coccolithophores would be changed on different levels. Thereby, potential changes in species distributions and abundances could propagate through multiple trophic levels of marine food webs. Although research into the long-term ecosystem impacts of ocean acidification is in its infancy, these results may indicate possible changes in biodiversity, trophic interactions, biogeochemical cycles, and other ecosystem processes.